Phosphoserine Phosphatase Is Expressed in the Neural Stem Cell Niche
and Regulates Neural Stem and Progenitor Cell Proliferation
ICHIRO NAKANO,a,bJOSEPH D. DOUGHERTY,cKEVIN KIM,dIVAN KLEMENT,eDANIEL H. GESCHWIND,f,g,h
HARLEY I. KORNBLUMb,d,g,h
Departments ofaNeurological Surgery,bPediatrics,cNeurology,dMolecular and Medical Pharmacology,
ePathology,fNeurology, andgPsychiatry and thehSemel Institute, University of California Los Angeles,
Los Angeles, California, USA
Key Words. Phosphoserine phosphatase • Neural stem cell • Brain • Development • Serine • Adult stem cells
Phosphoserine phosphatase (PSP) metabolizes the conver-
sion of L-phosphoserine to L-serine, classically known as
an amino acid necessary for protein and nucleotide syn-
thesis and more recently suggested to be involved in
cell-to-cell signaling. Previously, we identified PSP as
being enriched in proliferating neural progenitors and
highly expressed by embryonic and hematopoietic stem
cells, suggesting a general role in stem cells. Here we
demonstrate that PSP is highly expressed in periventricu-
lar neural progenitors in the embryonic brain. In the
adult brain, PSP expression was observed in slowly divid-
ing or quiescent glial fibrillary acidic protein (GFAP)-
positive cells and CD24-positive ependymal cells in the
forebrain germinal zone adjacent to the lateral ventricle
and within GFAP-positive cells of the hippocampal sub-
granular zone, consistent with expression in adult neural
stem cells. In vitro, PSP overexpression promoted prolifer-
ation, whereas small interfering RNA-induced knockdown
inhibited proliferation of neural stem cells derived from
embryonic cortex and adult striatal subventricular zone.
The effects of PSP knockdown were partially rescued by
exogenous L-serine. These data support a role for PSP in
neural stem cell proliferation and suggest that in the adult
periventricular germinal zones, PSP may regulate signaling
between neural stem cells and other cells within the stem cell
niche. STEM CELLS 2007;25:1975–1984
Disclosure of potential conflicts of interest is found at the end of this article.
Neural stem cells are multipotent cells of the embryonic and
adult brain that have the potential to produce multiple lineages
of mature neural cells. Neural stem cells have traditionally been
identified by retrospective assessment of their self-renewal and
multipotency using techniques such as the neurosphere assay [1,
2]. Neural stem cells are found in the embryonic brain, as well
as in the ventricular zone and in the subventricular zone of the
lateral ventricle and in the subgranular zone of the dentate gyrus
in the adult brain [3, 4].
Recent studies have described various spatial and temporal
differences in neural stem cells [5–7]. For example, in the
embryonic brain, stem cells are highly proliferative, giving rise
to neuronal, astrocytic, and oligodendroglial precursors at dif-
ferent stages of development. In contrast, the current model of
adult neurogenesis suggests that slowly cycling neural stem
cells in the subventricular zone (SVZ) give rise to rapidly
cycling transient amplifying progenitor cells, which then pro-
duce immature neuroblasts for integration into the nervous
system [8–13]. Thus far, it seems clear that, at least in the adult
brain, the slowly cycling stem cells are glial fibrillary acidic
protein (GFAP)-positive [14, 15], although GFAP-positive cells
do not exist in the early embryonic brains . Furthermore,
even in the adult brain, clearly not all GFAP-positive cells, or
even the majority of them, are stem cells.
Both cell-extrinsic and cell-intrinsic factors have been
shown to influence the maintenance and regulation of the neu-
rogenic system in vivo [17, 18]. To identify further candidate
factors, we used a strategy of stepwise screening using stem
cell-containing culture systems in vitro [19–21]. We performed
gene expression profiling on cultures of neural and non-neural
stem cells to identify genes enriched in neural stem cells. We
further stratified these in vitro candidates by examining the
regions in vivo of their expression to confirm their presence in
stem cell-containing regions. A few genes, including phospho-
serine phosphatase (PSP), were remarkably enriched in germinal
zones of neurogenesis.
PSP has been characterized in various organisms for over 20
years as the enzyme that metabolizes the conversion of L-
phosphoserine to L-serine, and it is the rate-limiting enzyme in
the primary serine synthesis pathway [22–25]. No protein phos-
phatase activity has ever been reported for PSP. L-Serine is an
amino acid necessary for protein and nucleotide synthesis, as
well as potentially playing a role directly in regulating neuronal
differentiation. D-Serine, glycine, and L-phosphoserine have all
been shown to bind as cofactors or ligands to various extracel-
Correspondence: Harley I. Kornblum, M.D., Ph.D., Room 1126 CIMI, 700 Westwood Plaza, Los Angeles, California 90095, USA.
Telephone: 310-794-7866; Fax: 310-206-8975; e-mail: firstname.lastname@example.org; Daniel Geschwind, M.D., Ph.D., Department of
Neurology, UCLA School of Medicine, Box 951769, Gonda 2506A, Los Angeles, California 90095-1769, USA. Telephone: 310-206-6814;
Fax: 310-267-2041; e-mail: DHG@ucla.eduReceived January 18, 2007; accepted for publication April 20, 2007; first published online in
STEM CELLS EXPRESS May 10, 2007. ©AlphaMed Press 1066-5099/2007/$30.00/0 doi: 10.1634/stemcells.2007-0046
TISSUE-SPECIFIC STEM CELLS
lular receptors [22–25]. All three molecules are within one step
metabolically of PSP.
The purposes of this study were to determine the PSP-
expressing cell types in brain throughout development and to
determine the function of PSP using in vitro neural progenitor
cultures. In the brain, we identified high levels of PSP expres-
sion in neural stem or progenitor cells throughout brain devel-
opment: it is highly enriched in cells in the germinal zone lining
the ventricles during embryonic stages and in the GFAP-posi-
tive cells in neurogenic regions in the adult brain. Functional
studies with overexpression and knockdown of PSP demon-
strated both intrinsic and extrinsic roles for PSP in proliferation
of neural stem cells from both embryonic and adult brains.
These data suggest that PSP is expressed in the neural stem cell
niche and may regulate proliferation of neural stem cells.
MATERIALS AND METHODS
All experiments were performed using embryonic and adult CD1
mice obtained from the Jackson Laboratory (Bar Harbor, ME,
http://www.jax.org) and with the approval of UCLA’s Animal Re-
search Committee following NIH guidelines.
In Situ Hybridization
Hybridization was performed essentially as described . Probes
of two different fragments of PSP (accessions) or full-length PSP
yielded the same results. Sense controls showed no labeling in all
Development of Antibody
PSP antibody was produced by immunizing two New Zealand
White rabbits with synthetic peptide to the C terminus of the protein
(WYITDFVELLGELEE) conjugated to KLH. Western blot identi-
fied specific antisera that recognized an appropriately sized 25-kDa
band that was absent in preimmune serum and peptide-blocked
antisera. Antibody was then purified by affinity column (Sigma-
Genosys, St. Louis, http://www.sigmaaldrich.com).
Postnatal CD1 mice were perfused transcardially with ice-cold
phosphate-buffered saline (PBS) followed by ice-cold 4% parafor-
maldehyde in PBS, pH 7.4. Brains were removed, fixed in 4%
paraformaldehyde overnight, sunk in 20% sucrose PBS, frozen in
4-methylbutane, and stored at ?80°C until use. Sections (40 ?m)
were cut on a cryostat and stored in PBS 1% azide at 4°C until use.
Embryonic mice, 17 days postconception, were dissected from
timed pregnant CD1 dams, and fixed in 4% paraformaldehyde for
10 days. They were then embedded in 2% low melting point agarose
and cut on a Vibratome (Vibratome, St. Louis, http://www.
vibratome.com) instrument into 100-?m sections and stored in PBS
1% azide until further use.
Free-floating sections were incubated overnight in 24-well
plates on a rotator at room temperature in the presence of 0.1%
azide, 0.25% Triton, and 5% normal goat serum in 500 ?l of PBS
and primary antibody at the following concentrations: anti-?III
Tubulin (TuJ1), 1:1,000 (MMS-435P; Covance, Princeton, NJ,
http://www.covance.com); anti-mammalian achaete-scute homolog
1 (anti-MASH1), 1:20 (556604; BD Pharmingen, San Diego, http://
www.bdbiosciences.com/pharmingen); anti-proliferating cell nu-
clear antigen (PCNA), 1:10,000 (M 0879; DakoCytomation,
Glostrup, Denmark, http://www.dakocytomation.com); anti-5-
bromo-2?-deoxyuridine (BrdU), 1:5,000 (PAB105P; Maine Biotech-
nology, Portland, ME, http://www.mainebiotechnology.com); anti-
PDZ-binding kinase (anti-PBK) monoclonal, 1:50 (612,170; BD
Transduction, San Jose, CA, http://www.bdbiosciences.com); anti-
GFAP, 1:1,000 (AB1540; Chemicon, Temecula, CA, http://www.
chemicon.com); anti-PSP, 1:500; anti-CD24, 1:1,000, with no
Triton (sc-19651). 3-Phosphoglycerate dehydrogenase (3PGDH),
1:1,000, was a gift of Dr. Watanabe ).
For BrdU and PCNA, antigens were retrieved by incubating
sections for 1 hour at 65°C in 50% formamide, 2? SSC, and 30
minutes in 2.0 N HCl at 37°C. Secondary antibodies were diluted
1:1,000 and included cy2-, cy3-, and cy5-conjugated antibodies
(Jackson Immunoresearch Laboratories, West Grove, PA, http://
www.jacksonimmuno.com) and Alexa 350-, 488-, 568-, and 594-
conjugated antibodies (Molecular Probes Inc., Eugene, OR, http://
probes.invitrogen.com). In all cases, no primary controls yielded no
For double labeling with 3pgdh and PSP, PSP labeling was
performed as described above with Alexa 568-conjugated anti-
rabbit secondary antibody, and then tissue was blocked for 30
minutes in PBS with 5% normal goat serum and 5% normal rabbit
serum. 3pdgh was then incubated for 4 hours at 1:1,000 in the same
buffer, followed by 30 minutes in Alexa 488 anti-rabbit secondary.
No 488 signal was seen when the second primary was omitted, and
PSP-positive puncta in hypothalamus were always negative for
Alexa 488, demonstrating the efficacy of the blocking steps.
Nuclei were counterstained with 4,6-diamidino-2-phenylindole-
containing mounting medium (Vector Laboratories, Burlingame,
CA, http://www.vectorlabs.com) or with Topro-3-iodide (Molec-
ular Probes), a nuclear stain fluorescing in the far red range (650
nm), by exposing tissue sections for 5 minutes to a 20 ?M
solution in PBS.
All fluorescent images were acquired on either a Leica TCS-SP MP
confocal and multiphoton inverted microscope (Heidelberg, Ger-
many, http://www.leica.com) and a two-photon laser setup (Spectra-
Physics, Irvine, CA, http://www.newport.com/spectralanding) or a
Zeiss LSM 510 META confocal microscope (Carl Zeiss, Jena,
Germany, http://www.zeiss.com), using lasers and filters appropri-
ate for the fluorophores, and pseudocolored images were overlaid
with Zeiss software or Adobe Photoshop (Adobe Systems Inc., San
Jose, CA, http://www.adobe.com). Infrared wavelengths were most
often pseudocolored blue. To confirm colocalization of antibodies,
stacks of ?1-?m-thick high-power confocal images were acquired
and examined both as three-dimensional reconstructions and as
animations moving through the z-plane with the Zeiss LSM image
Immunocytochemistry of adherent progenitors was performed as
described previously [20, 27], using the following antibodies: TuJ1
(1:500; Berkeley Antibodies, Covance, http://www.crpinc.com) and
O4 (1:50; Chemicon). Primary antibodies were visualized with
Alexa 568-conjugated (red) and Alexa 488-conjugated (green) sec-
ondary antibodies (Molecular Probes). Hoechst 33342 (blue) was
used as a fluorescent nuclear counterstain.
Neural Progenitor Cultures
Neurosphere cultures were prepared as described previously .
Briefly, cortical telencephalon was removed from E12 CD1 mice,
and cerebral cortex was isolated from older animals (Charles River
Laboratories, Wilmington, MA, http://www.criver.com). Cells were
dissociated with a fire-polished glass pipette and resuspended at
50,000 cells per milliliter in Dulbecco’s modified Eagle’s medium
(DMEM)/Ham’s F-12 medium (Invitrogen, Carlsbad, CA, http://
Gaithersburg, MD, http://www.gibcobrl.com), 20 ng/ml basic fibro-
blast growth factor (bFGF) (Peprotech, Rocky Hill, NJ, http://www.
West Sacramento, CA, http://www.gembio.com), and heparin
(Sigma-Aldrich). Growth factors were added every 3 days. For
differentiation, culture medium was replaced into Neurobasal me-
dium (Invitrogen) supplemented with B27, 2% fetal bovine serum
(FBS), and 10-6M of all-trans-retinoic acid (Sigma-Aldrich), on
poly-L-lysine (PLL)-coated dishes, and maintained for up to 5 days.
For secondary sphere formation assay, the primary spheres were
dissociated and plated into 96-well microwell plates in a 0.2-ml
PSP in Neural Stem Cells
volume of growth medium at 40,000 cells per milliliter, and the
resultant sphere numbers were counted at 7 days.
To assay the influence of gene knockdown or overexpression,
the neurosphere culture system was modified. Neurospheres were
propagated for 1 week and then dissociated with trypsin (0.05%)
followed by trituration with a fire-polished pipette. The cells were
then placed in DMEM/Ham’s F-12 medium with 2% FBS (Gibco-
BRL) and plated onto polyornithine/fibronectin-coated glass cover-
slips . After 6 hours, the serum-containing medium was re-
moved and the cells were placed back in the neurosphere growth
medium without heparin and supplemented with bFGF (20 ng/ml).
Transfection was then performed as described below. To assay the
sphere-forming potential of the transfected cells, they were lifted off
the plate with trypsin (0.05%), incubated briefly in medium con-
taining 10% FBS to inactivate trypsin, spun, and then placed into
neurobasal medium supplemented with B27, bFGF, and heparin. To
assay the function of cells expressing enhanced green fluorescence
protein (EGFP) driven by the maternal embryonic leucine-zipper
kinase (MELK) promoter, neurospheres at 7 days in vitro (DIV)
were plated onto coverslips as described above and transfected.
Some cultures were then placed into neurosphere conditions to
assay sphere-forming potential, whereas others were propagated and
differentiated on the coated coverslips after transfection.
GFAP-Positive Astrocyte-Enriched Cultures
Primary astrocyte cultures were prepared from P1 mouse cortices as
described previously . Briefly, as cells became confluent (12–14
DIV), they were shaken at 200 rpm overnight to remove nonadher-
ent cells and obtain pure astrocytes and passaged onto PLL-coated
coverslips for RNA collection or fibroblast growth factor stimula-
Reverse Transcription Polymerase Chain Reaction
Total RNA was isolated from each sample using TRIzol (Gibco-
BRL), and 1 ?g of RNA was converted to cDNA by reverse
transcription following the manufacturer’s protocol (Impron, Pro-
mega, Madison, WI, http://www.promega.com). For semiquantita-
tive reverse transcription-polymerase chain reaction (RT-PCR), the
amount of cDNA was examined by RT-PCR using primers for the
glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) gene as an in-
ternal control from 20–40 cycles. After correction for GAPDH
signal for each set, the resultant cDNA was subjected to PCR
analysis using gene-specific primers listed in supplemental online
Table 1. The protocol for the thermal cycler was as follows: dena-
turation at 94°C for 3 minutes, followed by cycles of 94°C (30
seconds), 60°C (1 minute), and 72°C (1 minutes), with the reaction
terminated by a final 10-minute incubation at 72°C. Control exper-
iments were done either without reverse transcriptase and/or with-
out template cDNA to ensure that the results were not due to
amplification of genomic or contaminating DNA. Each reaction was
visualized after 2% agarose gel electrophoresis for 30 minutes, and
expression levels were compared between the cDNA samples on the
same gel. PSP primers were as follows: sense, 5?-ccaggaaccgcggag-
gaaaactt-3?; antisense, 5?-cggctgtcggctgcatctcatc-3?. Sequences of
other genes will be provided upon request.
Flow Cytometry and Sorting
Flow cytometry and sorting with LeX antibody  and propidium
iodide were performed with a FACSVantage flow cytometer (Bec-
ton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.
com) using a purification-mode algorithm. Gating parameters were
set by side and forward scatter to eliminate dead and aggregated
cells. Background signals were determined by incubation of the
same set of progenitors without primary antibody. Isolated LeX?,
isolated LeX?, dividing (4n) cells, and nondividing (2n) cells were
dissolved in TRIzol for RNA purification. E12 progenitors were
labeled with LeX antibody (Invitrogen) for 30 minutes, and Alexa
530 was used for flow cytometry and sorting.
Cells were transfected using Lipofectamine 2000 (Invitrogen) fol-
lowing the manufacturer’s protocol. For transfection of plasmid
vectors, the cells were incubated with reagents for 6 hours with the
primary progenitor cells. For transfection of the double-stranded
siRNA complex, serial dilutions of siRNA from 5 to 200 nM were
tested to obtain specific knockdown of the gene of interest, and 100
nM was chosen as the concentration for functional study. Incubation
with siRNA complex was 6 hours with primary cells. For negative
control of plasmid transfections, EGFP expression vector driven by
the cytomegalovirus promoter was used. For negative control of
siRNA transfections, either mock, negative control siRNA (catalog
no. AM4611; Ambion, Austin, TX, http://www.ambion.com) or
luciferase siRNA (catalog no. 4627; Ambion) transfection was used.
Vector and siRNA Synthesis
The full-length coding region of mouse PSP was amplified by PCR
using mouse embryonic neurospheres as a template and subcloned
into pGEM-T Easy vector (Promega). After sequence verification,
the PSP fragment was subcloned into pCMV-Tag vector (Strat-
agene, La Jolla, CA, http://www.stratagene.com) at the NotI site.
siRNA was synthesized using the Silencer siRNA Construction Kit
following the manufacturer’s instructions (Ambion). Sequences for
double-strand RNA synthesis of PSP were as follows: antisense,
5?-aaattctgtggtgtggaggcctgtctg-3?; sense, 5?-aacctccacaccacagaatc-
PSP Expression Is Highly Enriched in Neural Stem
and Progenitor Cells Cultured from Embryonic
and Postnatal Cortices
During early development, neural stem cells in the ventricular
zone proliferate rapidly and give rise to neuronal progenitors.
We cultured these progenitor cells using the neurosphere
method  and compared the expression of PSP mRNA and
protein between proliferating neurospheres and sister cultures
differentiated by withdrawal of growth factors (Fig. 1A–1D).
For the analysis of the PSP protein, we raised antisera against a
peptide corresponding to the C-terminus of PSP. The anti-PSP
antibody was first tested by immunocytochemistry. When the
PSP-Flag fusion protein was expressed in cultured neural pro-
genitor cells, the signals of the PSP antibody colocalized with
those of anti-Flag antibody (supplemental online Fig. 1). West-
ern blot of protein from cultured neurospheres recognized a
25-kDa band that was blocked by preincubation with PSP pep-
tide (Fig. 1B). The PSP protein was abundant in proliferating
neurospheres, whereas differentiated cells expressed less PSP
protein. Protein samples from muscle and liver in mice at
postnatal day 0 were used as negative and positive controls for
the Western blot, respectively . These results were consis-
tent with mRNA expression in neural progenitor cells (Fig. 1B).
To determine whether PSP was universally expressed within
the cultures or was restricted to a more progenitor-like popula-
tion within neurosphere cultures, we used a cell surface anti-
body, LeX, to enrich neural progenitors from these cultures as
described previously [28, 31] (Fig. 1C). Neurosphere-initiating
neural stem cells in the embryonic cortices are exclusively
found in the LeX-positive fractions . Strikingly, PSP was
highly restricted to the LeX-positive neural progenitor popula-
tion, similar to other stem cell-enriched genes, such as MELK
and SOX2 [28, 32–34] (Fig. 1C). Next, we separated embryonic
neural progenitors into dividing (4n) and nondividing (2n) pop-
ulations and examined gene expression (Fig. 1D). Similar to the
results with the LeX antibody, SOX2 and MELK were enriched
in the dividing population within neural progenitor culture,
whereas Msi1 was detected in nondividing population. PSP
expression was restricted to the dividing population of embry-
onic neural progenitors. These data suggest that PSP mRNA and
Nakano, Dougherty, Kim et al.
protein are highly enriched and virtually restricted to prolifer-
ating neural progenitors in the developing central nervous sys-
Postnatal neurogenesis is different from embryonic neuro-
genesis, and the neural stem cells in the adult SVZ are a subset
of the GFAP-positive cells . These slowly cycling neural
stem cells in the SVZ give rise to rapidly cycling transient
amplifying progenitor cells, which then produce immature neu-
roblasts for integration into the nervous system [5, 6, 36]. To test
whether PSP could be involved in adult neurogenesis, we used
a cell culture system that simulates the transition of those cell
types in vitro (Fig. 1E–1G). GFAP-positive astrocytes from P0
cortices (?95%) were cultured and then were stimulated with
bFGF to produce GFAP-negative progenitors, followed by
TuJ1-positive neuroblasts  (Fig. 1E). Previously, we dem-
onstrated that this culture system is an in vitro model to reca-
pitulate adult neurogenesis in the SVZ . As was expected,
RT-PCR demonstrated that GFAP expression wanes and MASH1
expression waxes throughout this induction (Fig. 1G). PSP expres-
sion was strongly downregulated following the addition of bFGF.
These data suggest that PSP is expressed by GFAP-positive pro-
genitors rather than their daughter cells, such as rapidly amplifying
progenitors or committed neuroblasts.
PSP Is Expressed in the Germinal Zones
To validate our in vitro studies, we investigated PSP expression
during neural development using in situ hybridization and im-
munohistochemistry (Figs. 2, 5). In the embryonic brain, PSP
transcripts were strongly expressed along the ventricular wall
from E13 to P0, as well as the developing dentate gyrus of
hippocampus at E17 and P0 (Fig. 2A). Strong signal was also
observed in the embryonic liver (Fig. 2A). This PSP expression
in the developing liver was also detected at the protein level by
Western blot with the PSP antibody (Fig. 1B) . No specific
signal was detected using the PSP sense probe (data not shown).
Enriched expression of PSP in the developing germinal zones
was also observed using immunohistochemistry (Fig. 2B). The
PSP protein was detected primarily in the ventricular zone
region rather than in the TuJ1-positive cortical plate region (Fig.
2B). Collectively, these data demonstrate that PSP mRNA and
protein are expressed in the germinal zones of mouse embryonic
Function of PSP in Neural Stem (or Multipotent
Progenitor) Cells in the Embryonic Brain
Only neural stem cells (or other self-renewing, multipotent
progenitors), not lineage-committed progenitors, are capable of
forming new tripotent neurospheres under clonal conditions [1,
2, 37]. Therefore, the number of new neurospheres depends on
the neural stem cell population within neurospheres. By taking
advantage of this phenomenon, we directly tested PSP function
in neural stem cells in culture. Overexpression of PSP enhanced
Figure 1. PSP expression in neural stem and progenitor cell cultures
from embryonic and adult brains. (A): A schema showing self-renewing
S and their daughter cells, N. Markers are shown below each cell type.
(B): Reverse transcription-polymerase chain reaction (RT-PCR) (left
panel) and Western blot (right panel) analysis of PSP expression in NS
and DC from P0. GAPDH was used as an internal control for RT-PCR.
Protein samples from muscle and liver were used as negative and
positive controls, respectively, for the Western blot. (C): RT-PCR
analysis of progenitors derived from E12 telencephalon after separation
into LeX-positive and LeX-negative cell factions by fluorescence-acti-
vated cell sorting. GAPDH was used as an internal control. (D): RT-
PCR analysis of progenitors derived from P0 cortices after separation
into nondividing (DNA content ? 2n) and dividing (DNA content ? 4n)
cells by flow cytometry. (E): A schema showing the lineage of subven-
tricular zone stem and progenitor cells. (F): Experimental design. (G):
RT-PCR of cortical astrocytes derived from P0 cortices following ad-
dition of bFGF for the time period shown. GAPDH was used as an
internal control. Abbreviations: bFGF, basic fibroblast growth factor;
DC, differentiated neurospheres; GAPDH, glyceraldehyde-3-phosphate-
dehydrogenase; GFAP, glial fibrillary acidic protein; N, neuroblasts;
NCAM, neural cell adhesion molecule; NF, neurofilament; NS, prolif-
erating neurospheres; PBK, PDZ-binding kinase; PSA, polysialic acid;
PSP, phosphoserine phosphatase; S, embryonic neural stem cells;
TOPK, T-Lak cell originating protein kinase.
film autoradiograms demonstrating in situ hybridization using PSP
cRNA in sagittal sections of E13 and E15 whole bodies, E17 head, and
P0 brain. Scale bar ? 4 mm. (B): The left shows the location of the brain
section at E17 displaced in the photomicrographs. Green signal indicates
PSP-expressing cells, and red signal indicates TuJ1-positive neuroblasts
by immunohistochemistry. Abbreviations: E, embryonic; P, postnatal;
PSP, phosphoserine phosphatase.
PSP expression in the embryonic brain. (A): Bright-field
PSP in Neural Stem Cells
the clonal neurosphere forming capacity of neural stem cells
derived from E12 telencephalon (Fig. 3). A similar tendency
was obtained by experiments with P0-derived cells, although the
effect was not statistically significant (Fig. 3B). The effect of
knockdown was more prominent (Fig. 3C), as treatment with
PSP siRNA resulted in a reduction of the neurosphere forming
capacity of neural stem cells both in E12 and P0 cells in a
dose-dependent manner. For example, at P0, the reduction of
neurosphere formation was approximately 75% compared with
control siRNA-treated progenitors (Fig. 3C).
PSP Does Not Significantly Affect Differentiation
Potential of Neural Progenitors
Next, we assessed whether alteration of PSP expression affects
neural progenitor differentiation potential (Fig. 4). PSP siRNA
treatment did not clearly influence morphology or fate of both
E12 and P0 progenitors (data not shown). After transfection of
either control siRNA or PSP siRNA, P0 progenitor cells were
differentiated for 3 days and stained with three major cell type
markers: TuJ1 for neurons, GFAP for astrocytes, and O4 for
oligodendrocytes. As the majority of cells were stained with
GFAP, we focused our evaluation on potential changes in the
numbers of neurons and oligodendrocytes. Withdrawal of
growth factors resulted in the appearance of both TuJ1-positive
neurons and O4-positive oligodendrocytes (Fig. 4A). PSP
downregulation did not result in any significant shift of potential
for immature neural progenitors to give rise to cells in all three
lineages (Fig. 4B, 4C). These data suggest that PSP regulates
neural stem cell proliferation without significant effect on their
PSP Is Expressed in Ependymal Cells and SVZ
Astrocytes in the Adult Brain
We then examined whether PSP was expressed in adult neural
stem cells in vivo by immunohistochemistry. Like PSP mRNA,
the strongest PSP protein expression in the adult brain was
detected specifically in cells lining the entire ventricular system
(Fig. 5). However, by examining the PSP-expressing cells with
confocal microscopy, we determined that PSP was expressed in
dual layers (Fig. 5B). First, PSP was expressed in a population
of cells lining the ventricle but also in a second layer of cells
approximately 20 ?m more lateral. This dual-layer pattern was
unique to the lateral ventricles and was not seen in the other
ventricles. In addition to this labeling along the lateral ventri-
cles, PSP was also expressed in glia-like cells in the hippocam-
pal subgranular layer (supplemental online Fig. 2). In addition to
these neurogenic regions with strong PSP-labeled cells, faint
labeling was also occasionally detectable in glia in other re-
gions, including the corpus callosum, cortical white matter, and
strings of beaded puncta, suggesting axonal varicosities, in the
hypothalamus (data not shown).
Heterogeneous cell populations exist in the neurogenic re-
gion along the lateral ventricles, including GFAP-positive neu-
ral stem cells, PBK/T-Lak cell originating protein kinase
(TOPK)-positive rapidly amplifying progenitors , and TuJ1-
positive neuroblasts, as well as a lining of ependymal cells along
the ventricular surface. To determine whether the PSP-positive
cells lining the ventricles were ependymal cells, we examined
colabeling with CD24 using confocal microscopy . Indeed,
the PSP-positive cells lining all of the ventricles were CD24-
positive (Fig. 5C), whereas cells of the second, more lateral
layer found exclusively in the lateral ventricle were not CD24-
positive. In addition to ependymal cells, the subventricular zone
of the lateral ventricle contains GFAP-positive cells, at least
some of which are neural stem cells . To determine whether
PSP was expressed in GFAP-positive cells, we examined colo-
calization of GFAP and PSP. We found that the PSP-positive
cells in the lateral layer did contain GFAP-positive fibers, as did
the PSP-positive ependymal cells, albeit more dimly (Fig. 5G).
We also found that the PSP-positive cells in the subgranular
zone of hippocampus were GFAP-positive (supplemental online
Fig. 2), whereas GFAP-positive cells in other parts of the
hippocampus were not PSP-immunoreactive (not shown).
Other populations of cells within the SVZ include transient
amplifying progenitors that arise from the slowly proliferating
GFAP-positive cell and immature migrating neurons that arise
from the transient amplifying progenitors [5, 6]. To determine
Figure 3. In vitro function of PSP in neural stem and progenitor cells
in the embryonic brain. (A): Reverse transcription-polymerase chain
reaction after either overexpression or siRNA-induced knockdown of
PSP using neural progenitors derived from P0 cortices in NS and DC.
(B): Effect of PSP overexpression on NS formation in cells derived from
E12 tc and P0 cx. (C): Effect of PSP knockdown on clonal NS formation
of neural stem cells in E12 tc and P0 cx. Numbers in parentheses
indicate the final concentration of siRNA in the culture wells. (B, C):
Columns indicate the percentage change from EGFP-transfected cells ?
SEM. ?, different from controls, p ? .05; ??, p ? .001, analysis of
variance followed by post hoc t test. Abbreviations: cx, cortices; DC,
differentiated cells; E, embryonic; EGFP, enhanced green fluorescence
protein; GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; MELK,
maternal embryonic leucine-zipper kinase; NC, negative control siRNA;
NS, neurospheres; P, postnatal; PSP, phosphoserine phosphatase; tc,
Nakano, Dougherty, Kim et al.
whether PSP is expressed in the transient amplifying progenitor,
we double-labeled the PSP-positive cells with PCNA, MASH1,
and PBK/TOPK. We did not detect any PSP-positive cells
double-labeled with PCNA-positive rapidly cycling progenitors
(Fig. 5E). Likewise, MASH1 and PBK/TOPK, which are ex-
pressed in proliferating neuronal and glial progenitors of the
SVZ [40, 41], were not double-labeled with PSP (Fig. 5F;
supplemental online Fig. 3). Next, we tested double labeling of
PSP with TuJ1, which is expressed strongly in immature neu-
roblasts as well as more mature neurons. Interestingly, although
PSP was not expressed by the TuJ1-positive cells, the dual
layers of PSP near the lateral ventricle seemed to envelop the
bright TuJ1-positive immature neurons (Fig. 5D). Likewise, in
the dentate gyrus, PSP-positive cells were not TuJ1-positive but
adjacent to those cells (supplemental online Fig. 2). These
results are consistent with PSP being expressed in the GFAP-
positive, slowly cycling stem cells adjacent to immature neu-
rons. Collectively, our expression data provide strong evidence
that PSP is highly expressed in the rapidly dividing neural stem
cells in the embryonic brain and that in the adult, PSP is
expressed in slowly cycling, GFAP-positive SVZ cells, which
may be stem cells. PSP is also strongly expressed in ependymal
cells lining all the ventricles, including the lateral ventricles.
However, our data do not indicate whether or not these cells are
also stem cells.
PSP Regulates Proliferation of Adult
The data described above indicated that PSP is strongly enriched
in the GFAP-positive SVZ cells. Recent studies, including ours,
have demonstrated that GFAP-positive cells but not other cell
types in this region are neurosphere-initiating neural stem cells
[11, 15, 30, 14]. We tested PSP function in adult neural stem or
progenitor cells using neurosphere cultures from adult SVZ
(Fig. 6). Similar to the experiments with embryonic progenitors
(Figs. 3, 4), either PSP or control siRNA was transfected into
progenitors from adult SVZ, and neurosphere forming capacity
was examined under clonal conditions. As was the case in
embryonic cells, treatment of adult neural progenitors with PSP
siRNA resulted in downregulation of PSP compared with con-
trol siRNA-treated cells (Fig. 6A). Subsequently, treatment of
adult neural stem/progenitor cells with PSP siRNA resulted in a
diminished number of secondary neurospheres formed under
clonal conditions (Fig. 6B). These findings suggest that PSP
regulates not only embryonic but also adult neural stem/progen-
itor cell proliferation. Although the data described in Figure 4
indicate a lack of effect of PSP downregulation on postnatal
neural progenitor differentiation, we do not yet know whether
there is any role for PSP in the differentiation of adult progen-
L-Serine Production Is a Partial Mechanism of
Action of PSP in the SVZ Neural
The specific expression of PSP in putative neural stem cells in
vivo, as well as in the dual layer of expression suggest that
serine metabolism may play important roles cell intrinsically
and/or by providing an important component of the neurogenic
niche in the brain. We reasoned that if PSP regulates neural stem
cells through its activity as an enzyme in the L-serine synthesis
pathway, then it should be coexpressed with other members of
that pathway. Another member of the same pathway is the
enzyme 3PGDH, which is two steps upstream of PSP (http://
sion of 3PGDH has previously been examined in the mouse
brain [26, 42]. We examined coexpression of PSP and 3PGDH
(Fig. 7A). All PSP-positive cells were also positive for 3PGDH.
In contrast, 3PGDH expression seemed to extend well beyond
PSP-positive regions, and consistent with previous studies [26,
42], 3PGDH appeared to be robustly expressed in astrocytes
throughout the brain. These data suggest that L-serine metabo-
lism in general and PSP in particular may be important regula-
tors of stem cells and their niche.
To some extent, it is not surprising that decreasing the
expression of an enzyme involved in L-serine metabolism de-
creases proliferation in a very actively proliferating population
of cells. L-Serine plays a significant role in the synthesis of new
nucleotides and new proteins, both of which are required for cell
cycle progression . We next asked, does exogenous L-serine
rescue the decreased neural stem cell proliferation induced by
the PSP knockdown? To answer this question, siRNA-treated
adult neural stem/progenitor cells were stimulated to form
clonal neurospheres with or without L-serine. Addition of L-
serine in the medium showed a partial rescue in the number of
clonal neurospheres, suggesting that the effect of downregula-
tion of PSP was partially due to an autocrine lack of L-serine.
However, several lines of evidence suggest that this simple
explanation for the impact of PSP siRNA is incomplete. First,
the rescue by L-serine was only partial, suggesting other effects
of siRNA knockdown. Second, in vivo PSP was highly ex-
Figure 4. Lack of an influence of altering PSP expression on neural progenitor differentiation. (A): Immunocytochemistry of D progenitors after
transfection with either NC or PSP siRNA. Neurons were labeled with TuJ1 (green), and oligodendrocytes were labeled with O4 (red). Hoechst (blue)
was used for nuclear staining. Scale bar ? 50 ?m. TuJ1-positive cell populations (B) and O4-positive cell populations (C) were counted in UD and
neural progenitors after transfection. Graphs show the percentage of each in UD and D conditions. By t test: a, NC in UD versus PSP in UD, p ?
.9943; b, NC in UD versus NC in D, p ? .0001; c, NC in D versus PSP in D, p ? .5133; d, PSP in UD versus PSP in D, p ? .0001; e, NC in UD
versus PSP in UD, p ? .8435; f, NC in UD versus NC in D, p ? .0017; g, ND in D versus PSP in D, p ? .8411; h, PSP in UD versus PSP in D,
p ? .0038.Three independent experiments were performed for each condition. Abbreviations: D, differentiated; NC, negative control siRNA; PSP,
phosphoserine phosphatase; UD, undifferentiated.
PSP in Neural Stem Cells
Figure 5. Strong expression of PSP in adult subventricular zone astrocytes and ependymal cells. (A): In situ hybridization of PSP with coronal section of
adult mouse brain. (B): Schemata showing regions where images for (C–G) were taken. (C–G): Immunohistochemistry and confocal microscopy of third
(green)-positive ependymal cells. (D–F) In the neurogenic SVZ, PSP (red)-positive cells (D) surround Tuj1 (green)-positive neuroblasts and PCNA
(green)-positive (E) or MASH1 (green)-positive (F) proliferating progenitors. (G): The PSP (red)-positive cells contain GFAP-positive fibers (green).
Abbreviations: GFAP, glial fibrillary acidic protein; PCNA, proliferating cell nuclear antigen; PSP, phosphoserine phosphatase.
Nakano, Dougherty, Kim et al.
pressed not by the highly proliferative transient amplifying cell
but by the relatively slowly cycling GFAP-positive cells. This
suggests that PSP may also have a paracrine role in the regula-
tion of the neurogenic niche.
GFAP-positive SVZ cells express both 3PGDH and PSP. To
examine potential paracrine effects, conditioned medium from
PSP siRNA-treated astrocytes was used. Astrocytes from P0
cortices were transfected with PSP or control siRNA, and their
conditioned medium was added to neural progenitors in E12
telencephalon following dissociation. The number of secondary
neurospheres was reduced by the medium from PSP siRNA-
treated astrocytes, although the effect was not dramatic com-
pared with the direct knockdown in E12 progenitors (Figs. 3C,
7C). However, although the impact on the number of spheres
was small, the effect of conditioned medium derived from PSP
siRNA-treated GFAP-positive cells on the size of spheres was
much more dramatic.
PSP Expression in the CNS Germinal Zones
Previously, we performed extensive gene expression profiling
and candidate stratification for neural stem cell and stem cell-
associated genes [20, 21]. Here, we undertook studies to deter-
mine the potential role of PSP in the brain. Analysis of expres-
sion indicated that PSP is highly expressed in the germinal
zones of embryonic mice, whereas in the adult, PSP is expressed
by slowly dividing or quiescent cells that are situated adjacent to
more rapidly cycling cells.
In vitro data with PSP siRNA and overexpression support a
direct role for PSP in the proliferation of neural progenitors, in
cells. (A): Reverse transcription-polymerase chain reaction of PSP after
transfection of siRNA in NS cultures. Luciferase siRNA was used as a
control. GAPDH was used as an internal control. (B): Effect of PSP
knockdown on NS formation under clonal conditions after transfection
with siRNA. Columns represent the percentage of luciferase controls ?
SEM for three independent experiments for each condition. Abbrevia-
tions: GAPDH, glyceraldehyde-3-phosphate-dehydrogenase; NS, neu-
rosphere; PSP, phosphoserine phosphatase; SVZ, subventricular zone.
Function of PSP in the proliferation of adult neural stem
Figure 7. Autocrine and paracrine effects of PSP on neural stem/progenitor cell proliferation. (A): Coexpression of PSP (red) with another enzyme
in L-serine synthesis pathway, 3PGDH (green) in the anterior subventricular zone. (B): Effect of L-serine on clonal NS formation derived from adult
subventricular zone stem cells transfected with PSP siRNA. The graph shows the numbers of NS as a percentage of luciferase siRNA-transfected
controls. (C): NS counts (left) and diameters (right) from E12 progenitors cultured with conditioned medium of astrocytes transfected with siRNA.
Columns in (C) show the percentage of mock-transfected NS ? SEM. NS size is a histogram of the frequency of percentage of total NS found in
each size group. ?, different from controls, p ? .05, analysis of variance followed by post hoc t test. Counts for (B) and (C) are based on two
independent experiments for each condition. Abbreviations: 3PGDH, 3-phosphoglycerate dehydrogenase; NC, negative control siRNA; NS,
neurosphere; PSP, phosphoserine phosphatase.
PSP in Neural Stem Cells
a cell autonomous or autocrine fashion. Serine not only is
important as an amino acid but also can play a role in DNA
synthesis by serving as a substrate for purine biosynthesis .
Thus, it would be reasonable to presume that adequate expres-
sion of PSP would be required for optimal proliferation of
neural progenitors, especially under conditions where this pro-
liferation is being driven to its maximal extent, such as in the
neurosphere formation assay in vitro.
However, serine and phosphoserine may also play a role in
paracrine signaling. In the adult brain, PSP is not expressed by
rapidly proliferative cells, yet knockdown of PSP inhibits adult
neurosphere formation, suggesting that PSP expressed by rela-
tively quiescent cells influences either their own entry into the
cell cycle or the proliferation of more rapidly proliferative
progenitors. Conditioned medium derived from PSP siRNA-
treated astrocytes (GFAP-positive cells) inhibited neurosphere
formation and growth as compared with control conditioned
medium, suggesting that PSP regulates the production of a
substance that regulates proliferation. The target of this sub-
stance could be stem cells themselves, as suggested by the
effects on neurosphere number, and/or other proliferative pro-
genitors, as indicated by the effects on neurosphere size.
The latter observation—that conditioned medium derived
from PSP siRNA-treated cells has a greater effect on sphere
size, whereas direct siRNA treatment has a more profound effect
on sphere number, may seem somewhat contradictory. How-
ever, this points to a limitation of the methods used. Given our
estimated rates of transfection efficiency using this system
(65%; ), we reason that it is likely that PSP siRNA abolishes
sphere formation in virtually every sphere-forming progenitor
that is transfected. Thus, there would be no opportunity to
determine direct or indirect effects on other progenitors that
would have existed within the sphere and that would likely
influence sphere size.
The identity of the secreted substance regulated by PSP
could be serine itself. This is consistent with our observation
that addition of serine to the medium at least partially rescues
effects of PSP knockdown on neurosphere formation. The fact
that the rescue is partial could be related to methodological
issues, such as degradation of added serine or lack of access to
all cells, or it could indicate that PSP plays other important
roles. An intriguing possibility is that the function of PSP could
be to actually remove a substance that inhibits progenitor pro-
liferation. A recent screen of small molecule libraries identified
L-phosphoserine as one candidate that inhibited proliferation
and enhanced neuronal differentiation in embryonic progenitor
cells . Phosphoserine can bind on the N-methyl-D-aspartate
site of the NDMA receptor or act as an agonist for metabotropic
glutamate receptors [45, 46]. High expression of PSP in neuro-
genic regions may serve to eliminate this signaling molecule by
catabolizing phosphoserine to L-serine, regulating the level of
proliferation and differentiation in neurogenic regions of adult
In Vivo Function of PSP
The present study demonstrated that PSP plays a role in prolif-
eration of embryonic and adult neural progenitors, at least partly
through regulation of L-serine production. However, it is yet to
be determined whether PSP has the same role in vivo. The
absence of 3PGDH, the enzyme upstream of PSP, leads to
reduction of L-serine resulting in hypoplasia of the telencepha-
lon, diencephalon, and mesencephalon . Furthermore, both
patients with PSP deficiency and patients with 3PGDH defi-
ciency have impaired brain development, resulting in micro-
cephaly—an expected result if these enzymes regulate stem or
progenitor cell proliferation. In one patient with PSP deficiency,
treatment with oral serine led to normalization of serine levels
and some improvement in head growth [48, 49], a finding
consistent with a role for PSP in supplying L-serine. It will be
intriguing to examine the in vivo function of PSP for brain
morphogenesis during murine development and adulthood.
This work was supported by National Institute of Mental Health
Grant MH065756 and the Miriam and Sheldon Adelson Pro-
gram in Neural Repair Research. I.N. was supported by a
Institute for Stem Cell Biology and Medicine (ISCBM)-Califor-
nia Institute for Regenerative Medicine (CIRM) fellowship.
J.D.D. was supported by a Howard Hughes Medical Institute
(HHMI) predoctoral fellowship. The 3pgdh antibody was a gift
of Dr. Masahiko Watanabe (). I.N. and J.D.D. contributed
equally to this work.
DISCLOSURE OF POTENTIAL CONFLICTS
The authors indicate no potential conflicts of interest.
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PSP in Neural Stem Cells